Hiroshi Nikaido

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

SUMMARY Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Multidrug resistance in bacteria.

Large amounts of antibiotics used for human therapy, as well as for farm animals and even for fish in aquaculture, resulted in the selection of pathogenic bacteria resistant to multiple drugs. Multidrug resistance in bacteria may be generated by one of two mechanisms. First, these bacteria may accumulate multiple genes, each coding for resistance to a single drug, within a single cell. This accumulation occurs typically on resistance (R) plasmids. Second, multidrug resistance may also occur by the increased expression of genes that code for multidrug efflux pumps, extruding a wide range of drugs. This review discusses our current knowledge on the molecular mechanisms involved in both types of resistance.

Efflux-Mediated Drug Resistance in Bacteria

Drug resistance in bacteria, and especially resistance to multiple antibacterials, has attracted much attention in recent years. In addition to the well known mechanisms, such as inactivation of drugs and alteration of targets, active efflux is now known to play a major role in the resistance of many species to antibacterials. Drug-specific efflux (e.g. that of tetracycline) has been recognised as the major mechanism of resistance to this drug in Gram-negative bacteria. In addition, we now recognise that multidrug efflux pumps are becoming increasingly important. Such pumps play major roles in the antiseptic resistance of Staphylococcus aureus, and fluoroquinolone resistance of S. aureus and Streptococcus pneumoniae. Multidrug pumps, often with very wide substrate specificity, are not only essential for the intrinsic resistance of many Gram-negative bacteria but also produce elevated levels of resistance when overexpressed. Paradoxically, ‘advanced’ agents for which resistance is unlikely to be caused by traditional mechanisms, such as fluoroquinolones and β-lactams of the latest generations, are likely to select for overproduction mutants of these pumps and make the bacteria resistant in one step to practically all classes of antibacterial agents. Such overproduction mutants are also selected for by the use of antiseptics and biocides, increasingly incorporated into consumer products, and this is also of major concern. We can consider efflux pumps as potentially effective antibacterial targets. Inhibition of efflux pumps by an efflux pump inhibitor would restore the activity of an agent subject to efflux. An alternative approach is to develop antibacterials that would bypass the action of efflux pumps.

Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli

Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these β‐lactams across the outer membrane was not increased. This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi‐drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5mM decanoate. These results suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria‐Bertani medium. Finally, acrAB expression was shown to be increased in mar (multiple‐antibiotic‐resistant) mutants.

Efflux-mediated drug resistance in bacteria: an update.

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome, although they can also be plasmid-encoded. A previous article in this journal provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past 5 years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.

The outer membrane of Gram-negative bacteria.

Publisher Summary This chapter attempts to synthesize the current knowledge of the outer membrane, emphasizing the structural and molecular basis of various functions. The chapter explores that all the bacterial cells except those of mycoplasma and L-forms are surrounded by cell wall. It is a common knowledge that a fundamental difference exists in the structure of cell wall between the Gram-positive and Gram-negative prokaryotes, and that in the latter the cell wall contains an outer membrane layer in addition to the underlying peptidoglycan layer. The outer membrane especially that of Escherichia coli and Salmonella pphimurium, has been studied extensively during the last several years, and at present it claims to understand its structure and function properly at the molecular level. The chapter highlights that together with the erythrocyte membrane, the outer membrane is one of the best studied biological membranes.

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram‐negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram‐negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram‐positive bacteria, the inner membrane transporters from Gram‐negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two‐membrane envelope of Gram‐negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.

Outer membrane barrier as a mechanism of antimicrobial resistance.

La barriere de permeabilite, la membrane exterieure qui entoure les bacteries gram-negatives peut etre traversee par des solutes hydrophiles a travers les canaux porine. Ces canaux peuvent etre etroits et entraver la penetration de medicaments hydrophobes et creer differents degres de resistance intrinseque aux antibacteriens

The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals

Genes acrAB encode a multidrug efflux pump in Escherichia coli. We have previously reported that transcription of acrAB is increased under general stress conditions (i.e. 4% ethanol, 0.5 M NaCl, and the stationary phase in Luria‐Bertanl medium). In this study, lacZ transcriptional fusions and an in vitro gel mobility shift assay have been utilized to study the mechanisms governing the regulation of acrAB. We found that a closely linked gene, acrR, encoded a repressor of acrAB. Nevertheless, the general stress conditions increased transcription of acrAB in the absence of functional AcrR, and such conditions surprisingly increased the transcription of acrR even more strongly than that of acrAB. These results suggest that the general‐stress‐induced transcription of acrAB is primarily mediated by global regulatory pathway(s), and that one major role of AcrR is to function as a specific secondary modulator to fine tune the level of acrAB transcription and to prevent the unwanted overexpression of acrAB. To our knowledge, this represents a novel mechanism of regulating gene expression in E. coli. Evidence also suggests that the up‐regulation of acrAB expression under general stress conditions is not likely to be mediated by the known global regulators, such as MarA or SoxS, although elevated levels of these proteins were shown to increase the transcription of acrAB.

Antibiotic Resistance Caused by Gram-Negative Multidrug Efflux Pumps

Minimum inhibitory concentrations (MICs) of most lipophilic agents tend to be much higher against gram-negative than gram-positive bacteria. Multidrug efflux pumps that traverse both the inner and outer membranes make a major contribution to this intrinsic resistance of gram-negative bacteria. Such a pump is composed of at least three components, is energized by the proton-motive force, and can pump out not only an extremely wide variety of detergents, dyes, and antibiotics, but also those compounds, such as beta-lactams, that do not easily cross the cytoplasmic membrane. Increased expression of these pumps can raise the MICs to an impressive level. For example, 80% of carbenicillin-resistant clinical isolates of Pseudomonas aeruginosa from the British Isles owed their resistance to overexpression of an efflux pump and had carbenicillin MICs that were up to 2,000 times higher than that of the pump-deficient mutant strain.